In FY 2019 we have continued to develop and refine advanced methods for both quantitative measurement of signaling responses in macrophages (reviewed in Dorrington and Fraser (2019), Front Immunol. 10: 705) and for efficient gene perturbation in macrophage cell models (Lin et al, In preparation). We are also employing a Kinase Translocation Reporter (KTR) system for each subtype of the MAP kinases (Erk, p38 and Jnk) using spectrally distinct fluorescent proteins to permit the simultaneous dynamic measurement of activation of all three MAPK branches in macrophages. This technology has been introduced into cell lines and mouse models and promises to uncover a new appreciation of how MAPK dynamics are regulated in macrophages during inflammatory responses. Genetic screen data are susceptible to a myriad of experimental biases, some of which can be mitigated by computational analysis for which we have previously developed sophisticated software tools (Dutta et al (2016) Nat. Commun. 7: 10578). In FY 2019, we have further extended this work to complete the development of a novel bioinformatic method termed TRIAGE (Throughput Ranking from Iterative Analysis of Genomic Enrichment). This model applies multiple pathway and network enrichment steps on a screening dataset in an iterative manner, correcting for the biases of individual steps in a complementary fashion. To test and validate this approach, we analyzed a number of published HIV screens and observed a dramatic improvement in the ability to identify true positives at higher rates and the overlap rate between orthogonal screens of similar biology. We have developed a sophisticated web-based interface for the TRIAGE application, to permit its use by other investigators (https://triage.niaid.nih.gov). A manuscript describing the development and application of the TRIAGE application is in preparation. Using genome-wide screen data of the macrophage LPS response, we continue to characterize important cellular regulators of macrophage-driven inflammation and to publish collaborative studies related to our screen findings. In FY 2019 we published a study showing that the well-known host restriction factor for viral replication, IFIT1, has a role in reciprocally modulating the inflammatory and interferon gene programs in LPS-challenged macrophages (John et al, (2018) Cell Rep. 25: 95). Consistent with this finding, we have tested cells from patients carrying mutations in their IFIT1 gene and find a defect in type I IFN induction that may contribute to immune defects in these individuals. We believe that type I interferon and interferon stimulated genes have an under-appreciated role in regulation of host responses to bacterial infection. To further interrogate this question, in FY 2019 we initiated a CRISPR/Cas9 screen of ISGs for potential regulators of intracellular Gram-negative bacterial infection. Beyond our continued study of the TLR4 pathway response to bacterial LPS, we are also extending our studies to interrogate the recently discovered cytosolic LPS sensing pathway, which activates the non-canonical inflammasome response and the release of IL-1 family inflammatory cytokines. Recent studies have shown this to be a critical component of the broader physiological response to LPS and a major contributor towards septic shock outcomes in Gram-negative bacterial infections. We have collaborated with the NIH-NCATS screening facility to complete a genome-scale screen of the IL-1 alpha response to cytosolic LPS and we are actively studying the gene hits that have emerged from this project. In FY 2019, we have continued investigation of an important role for the mitochondria and cellular metabolism in inflammasome activation. Among genes emerging from our screen, we have identified three nucleotide diphosphate kinases, and we have further investigated the role of the Nme4 gene in inflammasome activation. We find that Nme4-/- RAW264.7 cells have a dramatic defect in their IL-1 alpha response to cytosolic LPS. They exhibit constitutively elevated cardiolipin levels in their mitochondrial outer membrane and show defective cardiolipin switching in response to mitochondrial stress signals. Interestingly, we find that Nme4-/- cells have a marked defect in the priming step of inflammasome activation, with a large majority of priming-induced transcriptional increases diminished in the absence of Nme4. Metabolic analysis suggests that Nme4 is critical to the glycolytic commitment induced during inflammasome priming, however we observe normal NF-kB and MAPK activation in primed Nme4-/- cells, suggesting that the mitochondrial and metabolic contribution to inflammasome priming occurs independently of these signaling responses. We also find that Nme4 deficient mice show substantial resistance to LPS-induced endotoxic shock. In ongoing studies, we are using dynamic live cell imaging reporters for mitochondrial function and inflammasome triggering, to further delineate the mitochondrial and metabolic processes that support inflammasome activation.